Method for detecting the biological activity of recombinant human growth hormone medicine

Abstract

The embodiments of the present disclosure provide method for Detecting the Biological Activity of Recombinant Human Growth Hormone Medicine. The result of biological activity detection method for GH medicine provided in present disclosure is stable and reliable. The operation is simple. The experimental period is short. there is no need for cells or other components from human primary tissue sources. The specificity and accuracy are high. The method can be used for product development and release inspection. It avoids cell contamination and errors caused by long-term incubation and multi-step operations. The color result is stable and the quality is controllable. The biological activity of GH medicine detected by the method is correlated with clinical efficacy, providing important reference for the treatment of related diseases in clinical practice.

What is claimed is: 
     
         1 . A method for constructing a cell line for stably detecting the biological activity of GH medicine, wherein said method comprises:
 a) transfecting plasmids stably expressing GHR, STAT5B, and 3H-IGF-1-P2-Luc reporter genes respectively into cells to obtain high titer lentivirus;   b) using the lentivirus stably expressing GHR and 3H-IGF-1-P2-Luc in step a) to infect target cell;   c) using the lentivirus stably expressing STAT5B in step a) to infect the target cell infected in step b) to obtain a cell line for stably detecting the biological activity of GH medicine;   the sequence of 3H-IGF-1-P2 is shown in SEQ ID NO: 5;   the target cell in step b) is HepG2 cell;   the vector for stable expression of GHR in step a) is vector pLVX-IRES-Hyg;   the vector for stable expression of STAT5B in step a) is vector Plentic-C-mGFP;   the vector for stable expression of 3H-IGF-1-P2-Luc in step a) is vector pLV-Puro.   
     
     
         2 . The method according to  claim 1 , wherein the plasmid for stable expression of GHR is pLVX-IRES-Hyg-Flag-GHR. 
     
     
         3 . The method according to  claim 1 , wherein the plasmid for stable expression of STAT5B is pLenti-MYC-STAT5B-IRES-BLAST. 
     
     
         4 . The method according to  claim 1 , wherein the plasmid for stable expression of 3H-IGF-1-P2-Luc is pLV-Puro-3H-IGF-1-P2-LUC2P. 
     
     
         5 . The method according to  claim 1 , wherein the cells in step a) are HEK293 cells. 
     
     
         6 . The method according to  claim 1 , wherein step a) further comprises the step of transfecting cells with plasmids for stably expressing GHR, STAT5B, and 3H-IGF-1-P2-Luc reporter genes which mix with plasmids pMD2.G and pSD, respectively. 
     
     
         7 . The method according to  claim 6 , wherein step a) further comprises the step of obtaining high titer lentivirus by ultracentrifugation after cell culture. 
     
     
         8 . The method according to  claim 1 , wherein the target cell after infection in step b) is obtained through screening. 
     
     
         9 . The method according to  claim 8 , wherein the target cells after infection are screened by antibiotics. 
     
     
         10 . The method according to  claim 1 , wherein the cell line for stably detecting the biological activity of GH medicine described in step c) is obtained through screening. 
     
     
         11 . The method according to  claim 10 , wherein antibiotics are used for screening. 
     
     
         12 . The method according to  claim 11 , wherein step c) further comprises screening by limited dilution method after antibiotic treatment. 
     
     
         13 . The method according to  claim 12 , wherein step c) further comprises washing and culturing the cells after the limited dilution method. 
     
     
         14 . A method for stably detecting the biological activity of GH medicine, wherein said method comprises:
 1) constructing a cell line stably expressing GHR, STAT5B, and 3H-IGF-1-P2-Luc reporter genes by the method according to claim  1 ;   2) coincubating the cell line in step 1) with a GH medicine sample;   3) adding luciferase substrate and determining the biological activity of the GH medicine.   
     
     
         15 . The method according to  claim 14 , wherein the GH medicine sample in step 2) is a sample of diluted GH medicine. 
     
     
         16 . A cell line for stably detecting the biological activity of GH medicine, wherein said cell line is constructed by the method according to  claim 1 . 
     
     
         17 . A product for stably detecting the biological activity of GH medicine, wherein said product comprises the cell line according to  claim 16 . 
     
     
         18 . The product according to  claim 17 , wherein the product further comprises diluent and luciferase substrate. 
     
     
         19 . The product according to  claim 17 , wherein the product further comprises instruction manual, positive control substance, and negative control substance. 
     
     
         20 . The product according to  claim 17 , wherein the product further comprises one or more sterile containers.